Project 1: Rac1 induces PCK-dependent myosinIIA phosphorylation to regulate association with focal adhesions and cell migration. Pasapera AM1, Fischer RS1,Plotnikov SV1,Egelhoff T2, Waterman CM1. Cell Biology and Physiology Center, NHLBI, NIH1;Department of Cell Biology, Lerner Research Institute NC-10, Cleveland Clinic.2 Cell migration requires coordinated assembly of focal adhesions and contraction in the actomyosin cytoskeleton. The small GTPase Rac1 is critical to cell migration through its known functions in regulation of focal adhesion and actin cytoskeletal assembly dynamics, but its role in regulation of myosin II is not known. Myosin II dynamically assembles into minifilaments at the leading edge of migrating cells, and PKC-mediated phosphorylation in Ser 1916 in the non-helical tail is one of the main regulators. We hypothesized that Rac1 may regulate myosin II minifilament assembly dynamics during cell migration via downstream regulation of PKC and Ser 1916 phosphorylation. To test this, we analyzed the effects of Rac1 activation on the phosphorylation and dynamics of myosin IIA in U2OS cells. We found that transfection of active Rac1 (Rac1V12) induced PKC- and integrin-dependent myosin IIA phosphoryation on Ser 1916. Live cell imaging of GFP-myosin IIA revealed that Rac1 activation promotes rapid assembly, motion, and turnover of myosin IIA minifilaments, as well as perpendicular orientation to the leading edge, resulting in its accumulation specifically in focal adhesions. To determine the role of Ser 1916 phosphorylation on myosin IIA dynamics and localization, we expressed phospho-mimetic (S1916D) and non-phosphorylatable mutants (S1916A) of myosin IIA. This showed that phosphorylation is critical to the Rac1-induced rapid assembly and turnover of myosin IIA minifilaments as well as to the focal adhesion association of myosin IIA. Thus, Rac1 acts as a master regulator of cell migration by coordinating actin assembly-mediated protrusion, adhesion, and actomyosin contraction dynamics. Project 2: PROTEOMIC ANALYSIS OF FOCAL ADHESION MATURATION Proteomic Analysis of Myosin II-mediated Focal Adhesion Maturation Reveals a Role for -Pix in Relaxation-mediated Rac1 Activation J. Kuo, X. Han, C.T Hsiao, J. Yates, C. M. Waterman Focal adhesions (FAs) undergo contraction-mediated maturation wherein they grow and change composition to differentially transduce signals from the extracellular matrix to modulate cell migration, growth and differentiation. To determine how FA protein composition is globally modulated by myosinII contraction, we developed a proteomics approach to isolate native FAs, identify their protein composition, and compare specific protein abundance in FAs from cells with and without myosinII inhibition. We reproducibly identified 905 FA-associated proteins, half (402) of which changed in FA abundance in response to perturbation of myosinII activity, thus defining the myosinII-responsive FA proteome. FA abundance of 75% of proteins in the myosinII-responsive FA proteome were enhanced by contractility, including those involved in Rho-mediated FA maturation, stress fiber formation, and endocytosis- and calpain-dependent FA disassembly. Surprisingly, 25% of the myosinII responsive FA proteome, including proteins involved in Rac-mediated lamellipodial protrusion, were enriched in FA by myosinII inhibition, establishing for the first time negative regulation of FA protein recruitment by contractility. We focused on the role of the Rac guanine nucleotide exchange factor, -PIX, documenting its depletion from FA during myosin-mediated FA maturation and its role in negative regulation of FA maturation to promote rapid FA turnover, lamellipodial protrusion and fast cell migration. This work was performed in collaboration with John Yates and Xuemei Han (Scripps) and C.T. Hsiao (U MD). Two manuscripts have been published on these studies Project 3: Analysis of traction stress variation across single focal adhesions. Sergey V. Plotnikov, Benedikt Sabass, Ulrich S. Schwarz, Clare M. Waterman The ability of eukaryotic cells to sense mechanical properties of the extracellular matrix (ECM) and to exhibit durotaxis (directed migration toward stiffer environments) is thought to underly many biological processes including angiogenesis, neurogenesis and cancer metastasis. ECM stiffness sensing is achieved by integrin-mediated focal adhesions (FA), protein assemblies that couple contractile actomyosin bundles to the plasma membrane and transmit force generated by the cytoskeleton to the ECM. Although it has been shown that both structural and signaling components of FA are crucial to translate mechanical cues into cell behavior, the molecular mechanism of mechanosensing remains unknown. Here we demonstrate that a molecular clutch, a mechanical link between the actin cytoskeleton and ECM-engaged integrins, acts as a mechanosensor in FAs, and the strength of the clutch determines range of ECM stiffness cells are able to sense to mediate durotaxis. Using high-resolution traction force microscopy on polyacrylamide ECMs of varying stiffnesses, we found that the mechanical behavior of the integrin-actin interface at FA exhibited ECM stiffness-dependent switching between a load-and-fail compliance sensing regime and a frictional slippage regime as described in the clutch oscillation model (Chan and Odde, 2008). In the load and fail regime, the position of peak traction within the FA resided on average at the distal FA tip, but oscillated over time towards the FA center and back to the tip. As ECM rigidity was increased, the traction peak did not oscillate and remained in the FA center, signifying the frictional slippage regime. We found that perturbing the gradient of paxillin phosphorylation across FA by expressing Y31/118E- or Y31/118F-paxillin mutants or by inhibiting FAK weakened the molecular clutch and switched FAs from load-and-fail compliance sensing to frictional slippage regime on compliant ECMs. In agreement with the clutch oscillation model, the load-and-fail regime could be rescued by further decreasing either substrate stiffness or myosin II contractility. Since paxillin phosphorylation on tyrosine residues 31 and 118 mediates vinculin recruitment into FAs, we demonstrated that vinculin and the paxillin-vinculin interaction are essential to strengthen the molecular clutch and to enable mechanosensing over a wide range of ECM compliances. We demonstrated the physiological importance of the load-and-fail compliance sensing regime by showing a requirement for this FA behavior in durotaxis, but not in chemotaxis in a boyden chamber assay or in random cell migration. This work was performed in colabboration with Benedikt Sabass and Ulrich Schwartz (university of Heidelberg) at the Marine Biological Laboratory in Woods HOle MA. It was presaented at the American Society for Cell Biology, Gordon conference on Signalling from Adhesion Receptors, and Frontiers in Cell Migration. A manuscript has been submitted on this work